Benzyl isothiocyanate is the chief or sole anthelmintic inpapaya seed extracts

Rohan Kermanshaia,1, Brian E. McCarryb, Jack Rosenfeldc, Peter S. Summersa,Elizabeth A. Weretilnyka, George J. Sorgera,*

aDepartment of Biology McMaster University, Hamilton, Ontario, Canada L8S 4K1bDepartment of Chemistry, McMaster University, Hamilton, Ontario, Canada, L8S 4K1cDepartment of Pathology, McMaster University, Hamilton, Ontario, Canada, L8S 4K1

Received 3 February 2000; received in revised form 22 December 2000

Abstract

Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on

silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and frac-tions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heat-treated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were

incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at roomtemperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate contentrelative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations andcyanogenic glucosides, which were present, could not account for the anthelmintic activity detected. Thus, our results are best

explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds areextracted. # 2001 Published by Elsevier Science Ltd. All rights reserved.

Keywords: Carica papaya; Caricaceae; Anthelmintic; Benzyl isothiocyanate; Caenorhabditis elegans; Myrosinase; Nematode

1. Introduction

Papaya seeds have been used for centuries as a ver-mifuge in India (Lal et al., 1976), Central and SouthAmerica (Roig y Mesa,1974) and throughout the world(Werner, 1992). Clinical trials with humans have led toseemingly contradictory results with Robinson (1958)claiming that papaya seeds are effective and Fernando(1959) claiming they are not. However, laboratory stu-dies have confirmed that various preparations of papayaseeds can kill helminths effectively in vitro and in infec-ted animals (Krishnakumari and Majumder, 1960; Daret al., 1965; Lal et al.,1976).The number and identity of anthelmintic compounds

present in papaya seeds has not yet been established.

Previous work has shown that seeds ground andextracted with either water or organic solvents, includ-ing alcohol, all produce crude extracts with anthelminticactivity and contain bioactive compounds such as ben-zyl isothiocyanate (BITC) (Dar et al., 1965; Ettlingerand Hodgkins, 1956; Krishnakumari and Majumder,1960; Tang, 1971; Tang et al., 1972). In some cases,diethyl ether could be used to concentrate the bioactiveprinciple(s) from water soluble seed extracts but, whilethe material that partitioned to the diethyl ether ororganic solvent layer was shown to have anthelminticactivity and contain BITC, fractions that were producedby the initial extraction of the seeds with water or whichpartitioned to the water layer were apparently nevertested for anthelmintic activity (Ettlinger and Hodgkins,1956; Tang, 1971, 1973). Steam distillates using water-soluble extracts of papaya seeds also yielded BITC andthe activity of this fraction against helminths wasdemonstrated (quoted in Ettlinger and Hodgkins, 1956).Since BITC is volatile and relatively insoluble in water,distillation of this compound from an aqueous solution

0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. All rights reserved.

PI I : S0031-9422(01 )00077-2

Phytochemistry 57 (2001) 427–435

www.elsevier.com/locate/phytochem

* Corresponding author. Tel.: +1-905-525-9140, ext. 24376; fax:

+1-905-522-6066.

E-mail address: sorger@mcmaster.ca (G.J. Sorger).1 Permanent address: Department of Biology, Faculty of Sciences,

Esfahan University, Esfahan, Iran.

at a relatively low temperature is not surprising. How-ever, the steam distillation carried out by these researcherswould not have identified any other putative bioactivecompounds that may have been heat-labile. Given thevariety of solvents and extraction conditions that canproduce extracts with bioactive properties against hel-minths, there is little compelling evidence that BITC isthe only anthelmintic principle that can be extractedfrom papaya seeds.The anthelmintic effect of papaya seeds has been var-

iously ascribed to carpaine (an alkaloid) and carpasemine(later identified as benzyl thiourea by Panse and Paranjpe,1943), and BITC (Krishnakumari and Majumder, 1960;Tang, 1971). Benzyl thiourea is reported to be an artifactthat arose during purification of the bioactive principledue to a reaction between ammonia and BITC (Ettlin-ger and Hodgkins, 1956) and it was present in prepara-tions from seeds at a concentration of one-tenth that ofBITC. Subsequently, Dar et al. (1965) tested BITC andbenzyl thiourea individually for bioactivity and showedBITC to be about 20 times more toxic to Ascaris lum-bricoides than benzyl thiourea. Since this study only tes-ted compounds already known to be present in papayaseed extracts it falls short of proving that BITC is theonly active anthelmintic principle in these seeds.In addition to anthelmintic properties, papaya seed

extracts also contain antimicrobial activity (Ettlingerand Hodgkins, 1956; Emeruna, 1982) due, at least inpart, to BITC (Das et al., 1954). Again, however, it is notclear whether all the antibiotic activities are due to one ormore compounds present in these seed preparations.In papaya seeds, BITC is formed from benzyl gluco-

sinolate (Gmelin and Kjær, 1970; Tang, 1973; Bennett etal., 1997), the major or perhaps only glucosinolate pre-sent. Glucosinolates in the seeds of many plants aremetabolized to yield isothiocyanates by the action ofenzymes commonly called myrosinases (thio-glucosidehydrolases, EC 3.2.3.1) (Ettlinger and Hodgkins, 1956;Palmieri et al., 1982), enzymes that are brought intocontact with their substrate(s) upon damage to seeds inwhich they are found. The myrosinase and glucosino-lates are in different compartments of the papaya seed(the endosperm and sarcotesta of the seed, respectively),although a small fraction of substrate and enzyme residetogether in the embryo (Tang, 1973). It follows thatpapaya seeds must be crushed or otherwise damaged toproduce substantial amounts of the antibiotic BITC.Different myrosinase/glucosinolate combinations give

rise to different products, with some producing a com-bination of thiocyanates and isothiocyanates, others amix of nitriles, thiocyanates and isothiocyanates andstill others isothiocyanates alone (Saarivirta and Virta-nen, 1963; Virtanen, 1965). It remains to be determinedwhether the myrosinase/benzyl glucosinolate combina-tion of papaya seed gives rise to all the possible pro-ducts listed above or only BITC.

More recently, Bennett et al. (1997) found thatpapaya leaves and stems contain cyanogenic glucosides.This finding was unanticipated because plants that con-tain glucosinolates do not, as a rule, contain cyanogenicglucosides (Bennett et al., 1997; Conn, 1980). However,cyanogenic glucosides offer a potential source of highlytoxic cyanide if they are also present in seeds. The pro-spect of cyanide contributing to the antibiotic propertiesof papaya seed preparations is difficult to address sinceseeds were not tested for these glucosides in the earlierstudy (Bennett et al., 1997) and so we felt it necessary totest for the presence of cyanogenic glucosides in papayaseeds.In this study we elected to use C. elegans as an end-

point to measure anthelmintic activity. While C. elegansis a free living nematode and not a parasite, it is con-sidered to be a good nematode model with importantgenetic similarities to parasitic nematodes (Blaxter,1998). Furthermore, parasitic nematodes have yet to bepropagated outside of their hosts. Our findings show thatanthelmintic activity is present or recovered from eitherwater-soluble (‘‘aqueous’’) or non-polar organic solvent(‘‘nonaqueous’’) extracts prepared from papaya seedsand that BITC is present in both extracts. We proposethat BITC is the major and probably only source ofanthelmintic activity present in papaya seeds and that it isproduced in preparations of these seeds primarily, andprobably exclusively, as the result of the action of residentmyrosinase(s).

2. Results

Commercial BITC (98% pure) was toxic to C. elegans,as expected (Table 1). Extracts of fresh papaya seeds pre-pared with water were also toxic to C. elegans and wereshown to contain BITC (Table 1). A volume of 10–20 ml ofthese aqueous extracts, when freshly prepared, represent-ing the contents of about 1.2–2.4 mg of seed, was sufficientto killC. elegans in our 0.5 ml assay. BITC was also foundin oil concentrates prepared from seeds that were Soxhletextracted with pentane and between 0.04 and 0.1 ml ofthis oil, representing the contents of 4–10 mg of seed,killed the nematodes in our assay (Table 1). If one con-siders the measure of toxicity as the LC90 or the con-centration of the compound needed to kill >90% of thenematodes within 4–5 h, this value is roughly equivalentregardless of the source of BITC (Table 1).Exposures for periods exceeding 12 h were generally

lethal to all the nematodes in the assay when the con-centration of BITC was at the LC90, irrespective of thesource of BITC. Fig. 1 shows the log of the concentra-tion of BITC in commercial BITC and in the aqueousand oil extracts of papaya seeds plotted against the logof the minimum volume of these preparations needed tokill 90% of the nematodes. A highly significant constant

428 R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435

relationship is found with an r2 of 0.989, an outcomethat is best explained if BITC is the active anthelminticprinciple in each of these preparations.When the seed residue, after having been thoroughly

extracted with pentane to remove BITC, is re-extractedwith water, the resulting extract is still a potent anthel-mintic (100 ml of this extract kills >90% of C. elegans).This could be explained if one or more of the activeprinciples present is soluble in water and not in pentane,or if a water-soluble precursor of the active principle isconverted to the active principle, that is only then solu-ble in pentane. Since benzyl glucosinolate is water solu-ble, is known to be present in papaya seeds (Tang, 1973;Bennett et al., 1997) and is a precursor of BITC that, inturn, is soluble in pentane, the latter hypothesis is plau-sible and could explain why both extractions yield BITC(Table 1).Myrosinase, an enzyme that catalyzes the conversion of

glucosinolates to isothiocyanates (Tang, 1973; Palmieri etal., 1982), was present in water extracts of ground papayaseeds and was found to elute in the void volume of a

Sephadex G-50 column, as expected, because of the highmolecular weight of myrosinases (Botti et al., 1995).Anthelmintic activity eluted much later from the columnand was absent from the void volume (results not shown).The results in Table 2 show that aqueous extracts of seedsground after heat-treatment of the seed exhibit no appar-ent nematode killing activity relative to extracts ofuntreated seeds at the volumes tested. Also, subjectingthe extract of heat-treated seeds to a cyclocondensationreaction with benzene dithiol (Zhang et al., 1992) pro-duced an absorption spectrum with no symmetricalpeak at 365 nm thus giving no evidence for the presenceof BITC (Fig. 2A). However, when extracts from theseheat-treated seeds were incubated with an aliquot of thevoid volume obtained from a Sephadex G-50 columnloaded with an extract prepared from fresh seeds, thenematode killing potential of the mixture increasedmarkedly (Table 2). When this preparation was subjectedto the cyclocondensation reaction described above, theabsorbance spectrum produced (Fig. 2B) is typical ofthat published for pure isothiocyanates processed in the

Table 1

BITC and papaya seed extracts decrease the viability of Caenorhabditis elegansa

Preparation Dose to kill>90% nematodes BITC

Volume (ml) Seed numberc Concentration (M) LC90d (mM)

BITC standardb 0.001–0.002 – 7.46 15.30

Seed extract Crude aqueous 10–20 0.03–0.06 0.0006 12–24

Crude oil 0.04–0.1 0.1–0.25 0.196 16.39

a Representative results shown, experiment was repeated three times.b Commercially available, 98% pure.c One seed weighs approximately 40 mg after the mucilaginous coat has been removed.d Concentration of BITC in the 0.5 ml nematode viability assay required to kill >90% of the C. elegans in 4–5 h.

Fig. 1. Regression plot showing the relationship between BITC con-

centration of various preparations and dose required to kill >90% of

Caenorhabditis elegans in a nematode killing assay.

Fig. 2. Absorption spectra of an extract of heat-treated seed before

(A) and after (B) incubation of the extract with a VoG50 fraction

containing myrosinase activity. For panel B, the sample was incubated

for 30 min and diluted eight fold with 10 mM potassium phosphate

(pH 6) buffer before measurement.

R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435 429

same fashion (Zhang et al., 1992). No nematode killingactivity was found in samples where the fresh seedextract obtained from the void volume of a SephadexG-50 column was boiled for 10 mm prior to incubationwith the heat-treated seed extract (Table 2). In a com-parable experiment the LC90 of BITC for the samplesubjected to activation conditions was determined to be20 mM. This LC90 is similar to that listed for all thepreparations used in Table 1, again suggesting thatBITC is the active nematode killing principle here.An oil fraction of papaya seed, prepared by Soxhlet

extraction with pentane, was applied to a silica columnand compounds present were separated by elution fromthe column with increasingly polar organic solvents.The BITC content and the C. elegans killing activityeluted in the same fraction (Table 3). In preliminaryexperiments, 40–60% of the nematode killing activity

eluted in a 90% hexane (H)/10% dichloromethane(DCM) mixture with the balance of the killing activityeluting in a 75% H/25% DCM mixture. The 90% H/10% DCM fraction was examined by GC and found tocontain a single prominent peak that upon subsequentexamination by mass spectroscopy and infrared spec-troscopy, was found to correspond to BITC (analysedby the Center for Mass Spectrometry, McMaster Uni-versity). The 75% H/25% DCM fraction was examinedby TLC, and upon exposure to iodine vapour, wasfound to contain a number of spots, so no further testswere performed with this fraction (results not shown).In subsequent fractionations, all the BITC and all theanthelmintic activity co-eluted from the column in amixture of 85% H/15% DCM (Table 3). Despite thepresence of other compounds as shown by TLC and arecovery of BITC that was less than 100% of the crude,this fraction yields an LC90 estimate which is similar tothat determined for the crude oil from which it wasproduced and one that lies within the range of valuesquoted in Table 1.We noted that the nematode killing power of aqueous

extracts of papaya seeds incubated at room temperaturefor 10 h decreased to 30–50% of a freshly preparedextract (Table 4). Aqueous extracts of papaya seedswere either extracted immediately with diethyl ether orfollowing a 10 h incubation at room temperature. Bothdiethyl ether extracts were then fractionated on a silicacolumn and the C. elegans killing activity and BITCcontent of each solvent fraction were determined. Forboth the unincubated and incubated samples, thenematode killing activity and BITC co-eluted in the85% H/15% DCM fraction (Table 4). In the case of thesample that had first been incubated for 10 h, the BITCcontent of the 85% H/15% DCM fraction was only halfthat found in the same fraction of the unincubated

Table 2

Loss of toxicity in aqueous papaya seed extracts due to heat treatment

can be restored upon incubation with a protein fraction prepared from

fresh papaya seedsa

Incubation mixture Dosage tested in

nematode viability test

Main componentb Additionc Volume

(ml)Nematodes

killed (%)

Buffer VoG50d 100 0

Extract of heat-treated Buffere 100 0

seed in buffer VoG50 50 100

100 100

Boiled VoG50 100 0

a Representative results shown, experiment was repeated five times.b 0.5 ml Volume with buffer comprised of 10 mM potassium phos-

phate (pH 6.0) and 1 mM ascorbic acid.c Volume of addition was 0.2 ml.d VoG50 is the void volume fraction from a Sephadex G-50 column

(see Experimental). No nematode killing activity was detected in this

fraction.e 10 mM potassium phosphate (pH 6.0) and 1 mM ascorbic acid.

Table 3

BITC content and toxicity of silica column fractions of papaya seed

extractsb

Solvent for elution of fraction BITC

Concentration

(mM)aLC90

(mM)

Crude oil 196 39

100% Hexane 0 0

85% Hexane/15% dichloromethane 130 29

100% Dichloromethane 0 0

100% Methanol 0 0

a The volume of each fraction was reduced by evaporation and, where

necessary, volumes were made equivalent by the addition of hexane.b Representative data shown. The experiment was completed twice

with identical results.

Table 4

Incubating crude seed extracts of papaya for 10 h reduces their BITC

content and toxicity to nematodes

Seed extract

incubationaFraction BITC

(mM)

Minimum volume

required for 100%

nematode kill (ml)c

None Crude n.d.b 30–50

85% Hexane/15% 504 16

dichloromethane

10 h Crude n.d. 100

85% Hexane/15% 240 50

dichloromethane

a Incubation at room temperature prior to chromatographic

separation of crude extract on silica and the subsequent testing for

BITC content and toxicity to nematodes.b n.d. Means not determined.c Volumes were corrected for differences in resuspension volume of

the chromatographic fractions (see Experimental for silica chromato-

graphy and GC quantification of BITC).

430 R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435

sample (Table 4). In separate experiments aliquots of theaqueous extract were incubated for up to 9 h at roomtemperature before extraction with diethyl ether. Thediethyl ether layer was then assayed for anthelminticactivity and BITC content. After six hours of incuba-tion, only 75% of the anthelmintic activity and 70% ofBITC remained relative to those measured for the non-incubated sample. After nine hours of incubation, thesevalues decreased to 50 and 39% for anthelmintic activityand BITC level, respectively. Thus, the same conclusionwas reached, C. elegans killing activity and BITC con-tent decreased with the length of incubation.In an effort to identify other putative anthelmintic

compounds in papaya seed extracts we attempted toobtain carpaine from the papaya seeds following the pro-cedure of Govindachari et al. (1965) but all our attemptswere unsuccessful. Seed extracts of Lepidium sativum havebeen found by colorimetric methods to contain BC (benzylcyanide) and BTC (benzyl thiocyanate) as well as BITC,their proportions varying with extraction conditions(Virtanen, 1962; Saarivirta and Virtanen, 1963, 1965;Gil and MacLeod, 1980). Preparations from Tropaeo-lum and Sinapsis alba seeds, on the other hand, con-tained only BITC (Saarivirta and Virtanen, 1963).Papaya seeds have not been examined specifically for

their content of BTC, BC and benzonitrile (BN). Anaqueous preparation of seeds was extracted twice withdiethyl ether, the diethyl ether layer was evaporated invacuuo and the resulting oil resuspended in hexane. Thissample, which contained all the anthelmintic activity fromthe extracted seeds, was applied to a silica column and theanthelmintic activity containing fraction (85% H/15%DCM), was subjected to GC under conditions that sepa-rate BITC, BTC and BC (Fig. 3, inset). BITC was foundas was an unknown compound tentatively identified asbutylated hydroxytoluene (BHT is introduced as thestabilizing additive for the diethyl ether) but no BTC orBC was detected in this preparation (Fig. 3). By an invitro assay, BITC was more potent as an anthelminticthan BTC and far more potent than BC (Table 5). Takentogether, these observations indicate that BTC and BC donot appear to contribute significantly towards the anthel-mintic power of papaya seed extracts. A commercialsource of benzonitrile (BN) was also examined for itstoxicity towards C. elegans and was found to have a veryhigh LC90 of >19 mM (Table 5). Also, BN has a reten-tion time of 2.98 min when subjected to the same GCelution conditions as used to separate BITC, BTC andBC (data not shown) and no such peak was noted onthe chromatograph shown in Fig. 3.Therefore, insufficient BN is present in the biologi-

cally active fraction for BN to be considered responsiblefor killing C. elegans.Bennett et al. (1997) reported that leaves and stems of

papaya seedlings contained cyanogenic glucosides withthe highest concentration found in leaves (extracts

showing 8 mM cyanide) but seeds were not tested. Wefound that the C. elegans LC90 for KCN exceeded 100mM in our killing assay (Table 5) and while cyanide atthis concentration caused nematodes to move moreslowly relative to the assay without cyanide, theyrecovered completely within 5 h. The cyanide content ofaqueous extracts of papaya seeds was always less than28 mM even after the extracts had been incubated withb-glucosidase at room temperature for two hours. Whenthese extracts are used in a nematode killing assay, thefinal concentration of cyanide present in the assay atwhich 90% of the nematodes are killed is less than 5mM, a concentration well below the 100 mM KCNfound to be non-lethal to nematodes. Processing apapaya seed sample with KCN added to a final con-centration of 20 mM yielded a sample whose concentra-tion was equivalent to that of the original sample plus

Table 5

Toxicity of various compounds to Caenorhabditis elegans

Compounda LC90 (mM)

BITC 15–45

Benzyl thiocyanate 200

Benzyl cyanide 5000

BN >19,000

KCN >100

a Commerically available compounds were dissolved in DMSO.

DMSO controls had no negative effect on nematodes.

Fig. 3. GC analysis of papaya seed preparations with anthelmintic

activity shows that BITC is present but BC and BTC are absent. An

aqueous papaya seed extract was extracted with diethyl ether, con-

centrated and dissolved in hexane and this was fractionated on a silica

column. The 85% H/15% DCM fraction containing anthehnintic

activity was evaporated to dryness and dissolved in CS2 for GC ana-

lysis (see Experimental). This experiment was performed twice with

similar results. The inset shows a GC separation of commercially

available BC, BTC and BITC.

R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435 431

the added KCN, demonstrating that cyanide was notdisappearing from the extract during the extractionprocedure. BITC added to extracts and to the cyanideassay mixture did not give rise to cyanide, hence therewas no complication from endogenous glucosinolates orBITC. It follows from the above that the cyanide/cya-nogenic glucoside contents of the papaya seed extractsdo not contribute to their anthelmintic power.

3. Discussion

3.1. BITC is the active compound in papaya seedpreparations

Our results show that the anthelmintic activity ofpapaya seed extracts prepared using water or pentaneco-purifies with BITC during column chromatography.Further, re-extraction of aqueous extracts with diethylether leads to a complete recovery of both bioactivityand BITC in the diethyl ether fraction. We noted thatchanges in nematode killing activity of preparations wereparalleled by changes in their BITC content. For example,the increase in anthelmintic activity obtained by incubat-ing extracts of heat-treated seeds with a soluble proteinfraction containing myrosinase activity is accompanied byan increase in BITC concentration. Also, when extractsare left for 10 h at room temperature, there is a decreasein killing activity of the extract that coincides with adecrease in BITC concentration. Aqueous extracts ofpapaya seeds do not appear to contain enough cyano-genic glucosides to be toxic to nematodes nor is thereany detectable BTC, BC or BN in the fraction contain-ing the anthelmintic activity. Taken together with theprevious literature, all these results indicate that the mainand perhaps the only effective anthelmintic in the papayaseed preparations we investigated is BITC. This conclu-sion supports the earlier proposal by Dar et al. (1965), butprovides a more thorough investigation of possible can-didate bioactive compounds naturally occurring in thepapaya seed extract.

3.2. Toxicity of papaya seed preparations

According to our measurements it takes the equiva-lent of 1.2–2.4 mg of seed to kill >90% of the C. ele-gans in our 500 ml assay within 4–5 h and completely ridthe assay mixture of live nematodes within 12 h. Giventhese in vitro viability results and assuming that thevolume of the small intestine of an adult human isapproximately 1.3 l (Masoro, 1973), then a dose of 3.1–6.2 g of papaya seeds would be equivalent to that whichis effective against C. elegans in our assay. However, thisdosage is likely to be an underestimate given that it doesnot consider factors that would reduce the intestinalBITC concentration over time including the movement

of contents through the gut [with the residence time of abolus being very short for the stomach and 3–7 h for thesmall intestine, (Osterwald, 1990; Fleisher et al., 1999)],and the likelihood that BITC is absorbed by the intest-inal epithelium. Our calculated dose approaches thepublished daily dose recommended for the medicinaluse of papaya seeds in Cuba (1–1.5 g three times a day,Roig y Mesa, 1974). If one gives the equivalent of 3.1–6.2 g of seeds to a 55 kg human, this amounts to a dose of56–112 mg/kg. Despite the speculative nature of thisdosage, this value was shown to be non-lethal to miceand rats (Chinoy et al., 1994) and a 5–10-fold lower dosewas shown to be effective against Ascaris in children(Robinson, 1958).A consideration relevant to the medicinal use of

papaya seeds, is that BITC must be released from ben-zyl glucosinolate by a papaya myrosinase. It followsthat factors that could inhibit myrosinase or inactivateBITC could compromise the concentration of BITC inthe small intestine. One such factor might be exposureto acid (pH 2) in the stomach (Magee, 1973). We testedthe effect of acid exposure on the potency of papayaseed extracts by deliberately acidifying aqueous seedextracts to pH 2.6 with HCl and then neutralized theextract to pH 6.5 with 2 M Na2CO3 either immediatelyor after one hour. Comparison of the in vitro anthel-mintic activity of these acidified and neutralized extractsrelative to that of an untreated control seed extractshowed that all the preparations were of equivalentpotency. An additional consideration that was notevaluated in this study is that there may be variabilityamong papayas with respect to myrosinase or glucosi-nolate content. Such variation or indeed any factor thatcould affect BITC content of seed extracts would, inturn, lead to differences in nematode killing potencybetween preparations.The BITC content of papaya seeds could be lethal to

some beneficial as well as harmful intestinal microorgan-isms (Emeruna, 1982). Thus while treatment with papayaseeds might eliminate intestinal bacteria, this treatmentwould not lead to an infection of the intestine by eukar-yotic parasites, such as Candida, since they would also bekilled by the BITC (Stoll and Seebeek, 1948).In addition to nematode killing activity, BITC

appears to elicit several biological effects. BITC inhibitschemically induced carcinogenesis in animal models andcell cultures (reviewed in Wattenberg, 1977; Zhang andTalalay, 1994) but is potentially toxic at therapeuticdosages since it is reported to be goitrogenic, carcino-genic and mutagenic (Yamaguchi, 1980; Fenwick et al.,1983). Thus a concern in any therapeutic application ofBITC must address how different are the therapeuticand toxic/lethal doses of the vermifuge.Fenwick et al. (1983) analysed data that suggests that

BITC is goitrogenic and proposed that the goitrogeniceffect is probably more associated with iodine deficiency

432 R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435

than to a direct effect of BITC alone. If such is the case,any adverse effects of BITC might be averted by aniodine supplement.Lohiya et al. (1994) showed that papaya seed extracts

administered to male rats for 30–60 days (at a doseequivalent to 67–333 mg/kg/day) can result in tempor-ary but completely reversible sterility. This daily dose iscomparable to our speculative dosage (see above), butthe comparatively short duration for therapy to killnematodes in humans should pose no threat in thisregard.Yamaguchi (1980) found that BITC at 50 mM

(roughly equivalent to 7.5 mg of papaya seed/l) doubledthe spontaneous reversion rate of Salmonella typhimur-ium strain TA100 and that activation by rat liver extractS-9 had no effect on this reversion rate. TA100 is muchmore sensitive than the wild type strain to mutagens anda petri dish assay is very different from the inside of amammalian intestine, but the 7.5 mg seed/l is onlyslightly greater than that used to kill C. elegans in ourassay (2.4–4.8 mg of seed/l) and could be, therefore, asource of concern. In contrast, at much higher con-centrations in experimental animals, BITC seems to actas an anticarcinogen that seems to be due to the antag-onistic effect of BITC on the enzymatic activation ofmutagens (Wattenberg, 1977; Zhang and Talalay, 1994;Talalay and Zhang, 1996; Fahey et al. 1997). Iso-thiocyanates, formed from their respective glucosino-lates, have been found in a number of different plants(notably in members of the Cruciferae) where they havebeen shown to have anticarcinogenic properties (Wat-tenberg, 1977; Zhang and Talalay, 1994; Talalay andZhang, 1996).With respect to cyanide/cyanogenic glucosides in

papaya seeds, a fatal dose of KCN for humans, accordingto Merck (1989), is between 1.85 and 2.22 mM and thefatal blood level of CN is reported as 115 mM (Henry,1979). These levels are obviously well above those foundby us in the seeds, let alone the levels expected after dilu-tion by the digestive tract, the bloodstream and inter- plusintrastitial water.It would appear from the foregoing discussion and from

many generations of traditional use of papaya seeds as ananthelmintic that this work, based upon chemical andbiological analysis of papaya seed extracts, supportsexisting practices and provides insight into how they maybe improved.

4. Experimental

4.1. Strains and materials

Caenorhabditis elegans strain N2 was obtained fromDr. J. Culotti, Hospital for Sick Children, Toronto,ON, Canada. BITC, 1,2 benzenedithiol, BTC, BC, BN,

N-chlorosuccinimide, succinimide, pyridine and barbi-turic acid were from Aldrich. Papaya (Carica papaya)was purchased locally.

4.2. Growth and maintenance of C. elegans and toxicityassay

C. elegans was maintained on solid NGM medium(Sulston and Hodgkin, 1988). Between 20 and 50nematodes were placed in 500 m1 of S medium (Sulstonand Hodgkin, 1988) in microtitre wells (Falcon No3047, Becton Dickinson) containing 30 ml of a 5 timesconcentrated overnight culture of E. coli WP2 (uvrA trpmalB, Witkin, 1975) resuspended in S medium. Aliquots(5–100 ml) of aqueous extracts (in water or 10 mMpotassium phosphate, pH 6) were added directly to thewells while concentrated oils, fractions in non-aqueoussolvents and commercial BITC were diluted with dime-thyl sulfoxide (DMSO) prior to being added to the wellsas 10 ml volumes. Dilute extracts prepared in non-aqu-eous solvents were concentrated first by evaporationunder N2, the residue resuspended with 1 ml of hexane,dried with air and the final residue was dissolved in 10 mlof DMSO which was then added to the test wells. Con-trols with the equivalent volumes of DMSO alone werenegative. Nematode survival was measured 4–5 h afteraddition of the extracts and again 12–13 h later.

4.3. Preparation of seed extracts

Seeds harvested from papaya fruit had their mucila-ginous coating removed prior to extract preparation.‘‘Aqueous extracts’’ were prepared by grinding seedswith a mortar and pestle using distilled water (1 g seedsto 10 ml water), at room temperature and removing thedebris by centrifugation at 16,000 g at 4�C for 30 min.‘‘Oils’’ were prepared by grinding seeds in a mortar andpestle, with no added solvent and then adding theground material to a thimble and extracting by refluxing10 times in 8 volumes of pentane, in a Soxhlet extractor.The final extract in pentane was concentrated in vacuuoat 38�C. A typical extraction of 14 g of seeds yieldedapproximately 0.9 ml of crude oil following Soxhletextraction.

4.4. Fractionation of extracts

Aqueous extracts (10 ml), prepared as describedabove, were extracted with 10 ml of diethyl ether andthe aqueous phase was recovered and re-extracted twomore times with 10 ml of diethyl ether each time. Thethree diethyl ether fractions were pooled and evapo-rated in vacuuo. The resulting residue was dissolved in 1ml of hexane and 0.45 ml was loaded onto a 10 g silica(Silica gel, 63–200 mm mesh, BDH) column (23�1.3cm). Oil prepared from seeds (described above) was also

R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435 433

fractionated by a comparable silica column. In this case,oil (0.45 ml) was loaded without further preparationonto the matrix. For fractionation of either aqueous oroil extracts, samples were eluted from the column at arate of 0.7 ml min�1 with successive 100 ml volumes of100% hexane, 85% hexane/15% dichloromethane,100% dichloromethane and 100% methanol. Theresulting eluates were evaporated in vacuuo at 42�C andthe residues were made to a final volume of 500 ml withhexane and stored at �18�C. Aliquots (10–100 ml) ofeach fraction were evaporated to dryness, the residuedissolved in 10 ml DMSO which was then used in thenematode killing bioassay to titre the potency of thefraction.

4.5. Activation of heated seed extracts with myrosinase

Seeds (with their mucilaginous coats removed) wereeither left unheated or were heat-treated by immersing atube containing seeds in boiling water for 10–20 mm orby autoclaving the seeds at 18 psi for 20 mm. Aqueousextracts were then prepared from the unheated andheat-treated seeds by grinding 1 g seeds with 10 ml of 10mM potassium phosphate (pH 6) buffer. One milliliterof extract from unheated seeds was loaded onto aSephadex G-50 column (10 ml bed volume). The frac-tion eluting in the 3.5 ml void volume (VoG50) con-tained the myrosinase activity (1.25 mmol sinigrindisappearing min�1 ml�1, using the assay of Palmieri etal., 1982). Heat-treated seed extracts (0.5 ml) wereincubated with 0.2 ml of the above VoG50 fraction for30 mm at room temperature and then placed on ice. l-Ascorbic acid was included at a final concentration of 1mM in the activation incubations. Aliquots of the acti-vated extract were removed and used to measure killingof C. elegans as described above (5–100 ml per assay)and measure BITC concentration (10–100 ml for colori-metric determinations or 1–5 ml of 10–50-fold dilutedpreparation for GC measurements).

4.6. Measurement and detection of compounds

TLC was used as an initial method for determiningpurity of extracts. Samples were spotted onto silicaGel-G plates, developed in methanol–n-butanol–conc.HCl–H2O (10:10:1:1; v/v/v/v), and plates were thenexposed to iodine vapours to visualize organic com-poundsColorimetric measurement of BITC was made fol-

lowing a procedure modified from that described byZhang et al. (1992). Two aliquots of a sample to bemeasured were each made to a final volume of 500 mlwith 10 mM Tris–HCl (pH 7.5). To one aliquot, 500 mlof 13 mM benzene dithiol in methanol was added whileto the second aliquot 500 ml of methanol (control) wasadded. A blank reaction with buffer and benzene dithiol

was also prepared. All of the reactions were incubatedat 65�C for 60 min in a microfuge tube and then micro-fuged for 5 min. Absorbance of the supernatant wastaken at 365 nm with a Uvikon 930 spectrophotometerand compared to those of a standard curve preparedusing commercial BITC.For BITC determinations, measurement using GC

was found to have greater specificity. GC was per-formed on a Hewlett Packard 5790A equipped with aflame ionization detector. The column was a MegaboreDBI column with a 30 m length, 0.52 mm inner dia-meter and 0.24 mm film thickness. The carrier gas wasH2 maintained at a flow rate of 12 ml min�1. Injectorport and flame ionization detector were set at 250 and300�C, respectively. Hydrogen and compressed air weredelivered to the detector at flow rates of 120 and 300 mlmin�1, respectively. Typical injection size was 1–3 ml ofsample dissolved in CS2 or diethyl ether. At injection,the oven temperature was 65�C and was maintained atthat temperature for 4 min and then increased at a rateof 5�C per min to a final temperature of 160�C. Theoutput signals from the detector were recorded andintegrated with a Shimadzu CR3A Chromatopac Inte-grator. Calibration using a dilution series of standardswith known concentrations was analysed prior to eachset of sample measurements. Concentrations weredetermined by comparing the responses from sampleswith those of the standards for injections of the samevolume. For GC analysis, papaya oil samples werediluted directly in CS2 or diethyl ether. Aqueous sam-ples were extracted into diethyl ether and either injecteddirectly into the GC or evaporated under N2 gas, theresidue dissolved in CS2 and then injected as above.Measurement of cyanogenic glucosides and cyanide

followed the procedure of Halkier and Møller (1989).Papaya seeds were ground with 50 mMMes-NaOH (pH6.5) and extracts incubated at room temperature withalmond b-glucosidase at 1 mg ml�1 (Sigma G-0395) for2 h. Reaction mixtures were adjusted to 0.83 M NaOHand incubated for 1 h to liberate free CN. A 50 ml ali-quot of glacial acetic acid was then added to each reac-tion mixture and the whole mixture was assayed for CNcontent (Halkier and Møller, 1989). The absorbance ofthe coloured complex formed was read at 585 and 650mm and the difference in absorbance values were usedto quantify CN levels by comparison to a standardcurve prepared with KCN.

Acknowledgements

We gratefully acknowledge K. Green, McMasterRegional Centre for Mass Spectrometry for mass spec-trometry analysis. This research was supported in partby a research grant to E.A.W. by the Natural Sciencesand Engineering Research Council of Canada.

434 R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435

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